1. Field of the Invention
This invention relates to a process for producing bacterial cells having a high content of acetate kinase. More specifically, it relates to a process for producing bacterial cells having a high content of acetate kinase per gram of dry cells, which comprises continuously cultivating thermophilic bacteria of the genus Bacillus under specified conditions.
2. Description of the Prior Art
In recent years, the utilization of enzymes as biochemical catalysts in areas not previously considered has been investigated in view of their often superior properties, such as (1) high specificity, (2) usefulness under mild reaction conditions, (3) energy conservation, and (4) freedom from pollution.
The trend of investigations of enzymes in the biochemical industry has shifted away from the earlier almost exclusive interest in hydrolases, and has more recently been directed to finding a way to reproduce energy sources for good efficiency and to utilize the advantages of enzymes in synthesizing compounds which are difficult to produce by chemical processes. To realize this, a so-called bioreactor has been developed. The bioreactor consists of a main reactor for producing substances, and a sub-reactor for supplying energy to the main reactor. The enzymes for use in the sub-reactor need to reproduce adenosine triphosphate (to be abbreviated ATP), which is a source of energy for living organisms, and the use of enzymes generically called phosphotransferases has been considered, and methods involving the use of phosphotransferases are known. For example, one method suggested comprises using glycerokinase (Journal of Applied Bacteriology, Vol. 38, pp. 301-304, 1975); a second method comprising the use of acetate kinase is described in Japanese Patent Application (OPI) No. 25088/77 (the term "OPI" as used herein refers to a "published unexamined Japanese patent application"); and a third method comprising the use of pyruvate kinase is described in Japanese Patent Application (OPI) No. 9392/78. Of these, the method using acetate kinase is considered to be most suitable because the equilibrium constant of the reaction during the production of ATP is very advantageous with respect to the production of ATP, and the acetylphosphoric acid source material required for the production of ATP by this enzyme is industrially available at low cost (American Institute of Chemical Engineers Journal, Vol. 22, p. 1079).
Since acetate kinase is an endobacterial enzyme, it is generally prepared by a method which comprises destroying the bacterial cells and extracting the enzyme from the resulting supernatant liquid. The supernatant liquid also contains various other substances derived from the bacterial cells, and it is extremely difficult to separate and purify the desired enzyme. For example, as described in Journal of Biochemistry, Vol. 84, No. 1, 195-203, 1978, acetate kinase can be purified by a complex process which involves destroying the bacterial cells, removing nucleic acids using Streptomycin, salting out the resulting supernatant liquid with ammonium sulfate, and subjecting the resulting crude enzyme precipitate to complex steps such as chromatography on a DEAE (Diethylaminoethyl) cellulose column, chromatography on a hydroxyappatite column, chromatography on an ultrogel column, and chromatography on a DEAE Sephadex column. Another complex method for purifying acetate kinase is described in Methods in Enzymology, Vol. I, pp. 591-595, including the steps of grinding up the bacterial cells and subjecting them to a combination of two acetone fractionations and three salting out steps with ammonium sulfate.
Because these methods are complex and timeconsuming, the cost of production of acetate kinase per unit of enzyme is very high, and this constitutes a serious obstacle to the industrial development of bioreactors.
Another problem in the production of acetate kinase is the low productivity of bacterial cells. Since acetate kinase is an endobacterial enzyme, the productivity would be increased generally by adding successively further nutrient sources to a fermentor tank in which cultivation is carried out by a batchwise method or by increasing the coefficient of oxygen transfer capacity by pressurizing the tank and/or passing oxygen, to thereby increase the amount of the bacterial cells. However, using such a cultivating method it is difficult to cause the logarithmic growth phase to continue, and so it becomes desirable to harvest cells which are about to shift to the stationary phase. Consequently, the content of the desired endobacterial enzyme is reduced, or a longer period of time is required for the cultivation. Furthermore, the cost of product is also increased, because of the increased consumption of the culture medium and oxygen. Thus, typically, although the amount of the cells that can finally be produced is increased, the amount of the desired cells per unit of time and per unit of cost of cultivation, and the productivity with respect to the enzyme, actually decreases. In fact, when these conventionally known techniques are applied to bacteria having the ability to produce acetate kinase, the content of acetate kinase in the bacterial cells is much smaller than that of bacterial cells which are cultivated by a batch process with reduced productivity of the cells and harvested during the logarithmic growth phase thereof.
Generally, enzymes participating in an energy metabolism system, such as glycerokinase or acetate kinase, are called constituent enzymes, and are known to be produced within bacterial cells irrespective of the cultivation conditions. For example, an article in the Journal of Applied Bacteriology, Vol. 38, pp. 301-304 (1975) stated that the productions of the rhodanase and glycerokinase as an endobacterial enzyme were investigated using a continuous cultivation method, and that the content of the enzyme per unit cell is dependent on the type of carbon sources in the nutrient medium, but is not affected by the dilution rate. An article in the Journal of Bacteriology, Vol. 133, No. 2, pp. 992-1001 (1978) stated that the relation between the content of acetate kinase per unit cells of Escherichia coli and its specific growth rate was investigated in a continuous cultivation method, and that no correlation was noted between the content of acetate kinase per unit cells and the specific growth rate. Furthermore, it is stated in Journal of Applied Chemistry and Biotechnology, Vol. 26, pp. 324-325 (1976) that the production of .alpha.-amylase by Bacillus stearothermophilus was investigated, and that the production of .alpha.-amylase as an exobacterial enzyme is proportional to the amount of bacterial cells. However, it is not described in the above Journal of Applied Chemistry that the productivity of .alpha.-amylase per unit cells is increased.